Preservation of blood platelets

ABSTRACT

Methods for preserving platelets at temperatures of less than about 15°C. with retention of hemostatic activity are provided. The method uses a first agent for inhibiting actin filament severing and a second agent for inhibiting actin polymerization. Contacting the platelets with the first and second agents prior to exposure to cold temperature prevents cold-induced platelet activation.

GOVERNMENT SUPPORT

This work funded by a government grant from the U.S. Public HealthService National Institute of Health, grant numbers HL19429 and HL47874.

FIELD OF THE INVENTION

This invention relates to methods and compositions for preservingplatelets at cryogenic temperatures with retention of hemostaticactivity. The methods involve the use of agents for inhibiting actinfilament severing and agents for inhibiting actin polymerization.

BACKGROUND OF THE INVENTION

The absence of adequate numbers of hemostatically active blood plateletsis associated with many disease states, some of which can only betreated by transfusion of blood products containing large numbers ofviable platelets. Freshly obtained blood platelets mediate hemostasis byconverting, where properly instructed, from discs to spiny pleatedspheres that attach to breaks in blood vessels and to other platelets.This process, referred to as platelet activation, is triggered by avariety of different agonists, including thrombin, adenosine diphosphate(ADP), thromboxanes, collagen, von Willebrand's factor, as well as uponcontact of platelets with glass.

Current practice permits platelets to be stored no longer than severaldays, after which the platelets are no longer hemostatically active andare discarded as "outdated". It is estimated that about 15% of procuredunits of blood are discarded as outdated. As a result of the shortplatelet shelf life, a large supply of donated blood is required tosustain each patient requiring platelet replacement therapy.

Given the problems of platelet availability, various attempts have beenmade to preserve platelets for longer periods of time with retention ofhemostatic activity. Most of this work was done in the 1960's and early1970's and culminated in the practice of room temperature storage. Thesestudies revealed that while room temperature storage led rapidly tosignificant reduction in hemostatic function, the phenomenon ofcold-induced platelet activation had more deleterious effects (Murphy,P. H. and Gardener, F. H., 1969 N. Engl. J. Med. 280:1094-1098; Handin,R. I. and Valeri, C. R., 1973 J. Engl. J. Med. 285:538-543). Morerecently, research has focused on the modification of platelet storagepacks or bags to increase porosity and gas exchange, on nutrients,metabolites, pH and protease inhibitors (e.g., Murphy, S. et al., 1982Blood 60:194-200; Rinder, H. M. and Snyder, E. L., 1992 Blood Cells18:445-456). Because storage at non-refrigerated temperatures has beenassociated with microbial contamination of transfused platelets(Bennett, J. V., 1971 N. Engl. J. Med. 285:457-458; Buckholz, D. H., etal., 1971 N. Engl. J. Med. 285:429-433; Morrow, J. F., et al., 1991 JAMA266:555-558) the Food and Drug Administration (FDA) limits plateletstorage to five days.

To date, efforts to store platelets at reduced temperatures have provenunsuccessful because of the morphological changes which plateletsundergo in response to cold temperatures. These changes, collectivelyreferred to as "cold-induced platelet activation", result insubstantially impaired hemostatic function. In contrast to freshlyobtained platelets, platelets that have been rewarmed followingcold-induced activation share many structural features withglass-activated platelets but have substantially impaired hemostaticactivity. Thus, although (agonist- or glass-induced) platelet activationand cold-induced platelet activation have in common some structuralsimilarities, these activation processes yield quite distinctivefunctional results. To understand the processes which compriseagonist-and/or cold-induced activation and the differences between thetwo types of activation, the cytoskeletal structure of the restingplatelet must first be considered.

Prior to activation, the resting platelet contains a highly organizedcytoskeletal structure, with actin representing about a fifth of thetotal protein (Hartwig, J., 1992 J. Cell Biology 118(6):1421-1442).About half of the actin in resting platelets is present as actin monomer("G-actin") and is stored as a 1:1 complex with beta4-thymosin orprofilin. The remainder of the actin in resting platelets is organizedinto long filaments ("F-actin") which radiate outwardly from theplatelet center. The filaments have a fast-growing end, the "barbedend", to which the actin monomers are added in a process alternativelyreferred to as actin assembly or actin polymerization.

Spontaneous actin assembly from monomers in vitro proceeds through athermodynamically unfavorable nucleation step that limits the initialrate of this polymerization reaction. In vivo, various proteins regulateplatelet activation by association with actin monomers and/or filaments.The presence in platelets of nearly stoichiometric quantities of actinmonomer binding proteins, e.g. profilin and beta 4-thymosin, withaffinities for actin monomer in the micromolar range, presumablyprevents spontaneous nucleation in vivo (Safer, D., et al., 1991 J.Biol. Chem. 266:4029-4032; Weeds, A. G., et al., 1992 Biochem. Soc.Trans. 19:1016-1020). By associating with actin monomers, these"sequestering proteins" render the monomers incapable of adding to thefree pointed ends of actin filaments and less capable of adding to the(uncapped) barbed ends of actin filaments.

The exact interplay of these regulatory proteins with actin monomers andfilaments and their involvement in platelet activation is not preciselyunderstood. In the resting platelet, actin filaments bind viaactin-binding proteins ("abp") to a dense spectrin-rich shell thatlaminates the plasma membrane (see e.g., Hartwig, J. and DeSisto, M.,1991 J. Cell Biol. 112:407-425). We have observed that upon stimulationby an agonist, such as thrombin, the resting platelet swells, presumablyas a result of actin filament severing (see Hartwig, J., 1992 supra.).It is known that severing requires an increase in the intracellular freecalcium concentration (Hartwig, J. and Yin, H. L., 1987 BioEssays7:176-179).

Exposure of platelets to thrombin increases the intracellular calciumconcentration to near micromolar levels in the absence of externalcalcium and to greater than micromolar levels when calcium is acomponent of the surrounding medium (see e.g., Oda, A., et al., 1991 Am.J. Physiol. 260:C242-C248). Calcium at micromolar levels leads to theformation of gelsolin-actin complexes in vitro (Stossel, T., 1989 J.Biol. Chem. 264:18261-18264). In the resting platelet, >95% of thegelsolin is free, i.e., not complexed to actin (Lind, et al., 1987 J.Cell Biol. 105:833-842). Free gelsolin (not gelsolin-actin complexes)reportedly plays a role in calcium-dependent actin filament severing(Janmey, P. A., et al., 1985 Biochemistry 24:3714-3723). Loading cellswith permeant calcium chelators reportedly quenches the increase inintracellular calcium concentration in response to agonists such asthrombin (Davies, T. D., et al., 1989 J. Biol. Chem. 264:19600-19606).

Various intracellular calcium chelating agents have been used asresearch tools to elucidate the role of calcium in platelet activation.These include derivatives and analogues of the calcium chelator BAPTAdeveloped by Tsien et al., (see e.g., U.S. Pat. No. 4,603,209). Many ofthese chelators exhibit an increase in fluorescence emission (inresponse to appropriate excitation) upon binding free calcium. However,to be useful as intracellular chelating agents, these calcium chelatorshad to be derivatized with lipophilic groups, i.e., to render thechelators capable of penetrating the platelet membrane and entering thecytosol. Such intracellular calcium chelators have been used to measureintracellular calcium concentrations in human blood platelets at restand during activation (Cobbold, P. and Rink, T., 1987 Biochem. J.248:313-328). Very low intracellular calcium concentrations wereachieved when large amounts of the chelators were loaded into thecytosol in the absence of an exogenous source of free (unchelated)calcium (Cobbold and Rink, 1987, supra.).

Platelet activation is manifested by transformation of the platelet intoa compact sphere from which extend spines (filopodia) and veils("lamellipodial networks") (FIG. 1). The filopodia comprise bundles ofactin filaments ("filopodial bundles"). The veils contain shorter actinfilaments and represent a second type of filament organization. Thegeneration of both of these actin structures requires gelsolin. Webelieve that removal of gelsolin from the core actin network, i.e., thepopulation of actin filaments deep within the platelet, leads toformation of the filopodial bundles and that removal of gelsolin fromsevered actin filaments leads to formation of the lamellipodial network.

Much of what is known about the structural changes accompanying plateletactivation has been learned from studying the barbed end actinpolymerization activity of detergent-demembranated platelets in variousstates of activation. Barbed end actin polymerization activity isdetermined by observing the rate at which newly added actin monomer isincorporated into platelet filaments (see e.g., Hartwig, J. and Janmey,P., 1989 Biochim. Biophys. Acta. 3030:64-71). Because cytochalasin B isa well known inhibitor of actin assembly onto the barbed ends of actinfilaments, the existence and extent of barbed end activity is determinedby observing the effect of cytochalasin B on the rate at which actinmonomers are added to the barbed ends of actin filaments.

The cytochalasins and the related chaetoglobosins constitute a class ofmore than 24 structurally and functionally related mold metabolites.Several publications have reported that cytochalasin B prevents some ofthe platelet shape changes associated with cold-induced activation, butthat other changes, e.g., distortions of intracellular membranes, werenot prevented (White, J. B. and Krivit, W., 1967 Blood 30:625; White, J.G., 1982 Am. J. Path. 108:184). More recently, the cytochalasins havebeen reported to alter actin-based cytoskeletal morphology (see e.g.,Schliwa, M., 1982 J. Cell Biol. 92:79-91) and inhibit actinpolymerization (see e.g., Mooseker, M. S., 1986 J. Cell Biol.102:282-288).

In vitro studies using purified actin indicate that cytochalasins bindto the barbed end of actin filaments and inhibit its polymerization (seee.g., Lin et al., 1980 J. Cell Biol. 84:455-460) by reducing the rate ofmonomer addition to the barbed end of growing filaments (see e.g.,Ohmori, H., et al., 1992, J. Cell Biol. 116(4):933-941 and referencescited therein). Although the detailed mechanism by which thecytochalasins inhibit actin polymerization has not been elucidated(e.g., Cooper, J. A., 1987 J. Cell Biol. 105:1473-1478), it is believedthat the cytochalasins and related compounds interfere with the dynamicequilibrium that exists in nonmuscle cells between actin filaments(F-actin) and monomeric actin (G-actin) (see e.g., Spector, I., et al.,1989 Cell Motility and the Cytoskeleton 13:127-144 and references citedtherein).

The above-cited references disclose the use of agents such ascytochalasin B and intracellular calcium chelators for characterizingthe biochemical and morphological changes that occur duringagonist-and/or glass-induced platelet activation. However, none of thecited references disclose the use of such agents, alone or incombination, for modulating or preventing cold-induced plateletactivation. Accordingly, there is still a need for methods andpharmaceutical compositions to preserve platelets. In particular, thereis still a need for methods for preserving platelets at cryopreservationtemperatures, which methods prevent cold-induced platelet activation.Such methods would permit the preservation of blood platelets withpreserved hemostatic activity for longer periods of time than arecurrently possible.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for thecryopreservation of platelets with preserved hemostatic activity. Alsoprovided are methods for making a pharmaceutical composition containingthe cryopreserved platelets and for administering the pharmaceuticalcomposition to a mammal to mediate hemostasis. The methods are basedupon the recognition by Applicants that two processes, actin filamentsevering and actin polymerization, are essential pathways involved incold-induced platelet activation and the concomitant loss of hemostaticfunction.

According to one aspect of the invention, a method for thecryopreservation of platelets with preserved hemostatic activity isprovided. The method comprises contacting a preparation of plateletswith a first agent for inhibiting actin filament severing and with asecond agent for inhibiting actin polymerization to form a treatedplatelet preparation and storing the treated platelet preparation at acryopreservation temperature. The platelets are collected fromperipheral blood by standard techniques known to those of ordinary skillin the art. In a preferred embodiment, the platelets are contained in apharmaceutically-acceptable carrier prior to treatment with the firstand second agents.

As used herein, actin filament severing refers to disruption of thenon-covalent crosslinks between actin filaments and between filamentsand the spectrin-rich shell that laminates the plasma membrane (FIG. 1).Severing requires an increase in the concentration of intracellularcalcium. Accordingly, in a preferred embodiment, the first agent is anintracellular calcium chelator that is capable of penetrating theplatelet membrane.

As used herein, actin polymerization refers to the process by whichactin monomers ("G-actin") are assembled onto the fast-growing ("barbedend") of actin filaments ("F-actin"). Exemplary second agents forinhibiting actin polymerization include a class of fungal metabolitesknown as the cytochalasins. It is believed that the cytochalasinsinhibit actin polmerization by constitutively mimicking the actions ofendogenous, metabolically regulated barbed end capping agents, e.g.,gelsolin, thereby reducing the rate of monomer addition onto the barbedend of growing filaments.

Following contact with the first and second agents, the treatedplatelets are stored at a cryopreservation temperature. As used herein,"cryopreservation temperature" refers to a temperature that is less thanabout 22° C. In a preferred embodiment, the cryopreservation temperatureis less than about 15° C. In a most preferred embodiment, thecryopreservation temperature ranges from between about 0° C. to about 4°C.

According to another aspect of the invention, a method for making apharmaceutical preparation for administration to a mammal is provided.The method comprises preparing the above-described cryopreservedplatelet preparation, warming the platelet preparation, and neutralizingthe first and second agents. If the treated platelets are not alreadycontained in a pharmaceutically acceptable carrier, they are placed in apharmaceutically-acceptable carrier prior to administration to themammal. As used herein, the terms "neutralize" or "neutralization" referto the process by which the first and second agents are renderedsubstantially incapable of further acting in the platelet preparation asagents for inhibiting actin filament severing and inhibiting actinpolymerization, respectively.

According to yet another aspect of the invention, a method for mediatinghemostasis in a mammal is provided. The method comprises administeringthe above-described pharmaceutical preparation to the mammal.

According to still another aspect of the invention, storage compositionsand pharmaceutical compositions for mediating hemostasis are provided.

In one embodiment, the compositions comprise a pharmaceuticallyacceptable carrier, a plurality of platelets, a plurality of a firstagent for inhibiting actin filament severing and a plurality of a secondagent for inhibiting actin polymerization. In a storage composition, thefirst and second agents are present in the composition in sufficientamounts so as to prevent cold-induced platelet activation. As usedherein, the phrase "cold-induced platelet activation" refers to themolecular and morphological changes that blood platelets undergofollowing exposure to cold temperatures, e.g., 4° C. In a pharmaceuticalcomposition, the agents have been neutralized and the compositioncomprises a pharmaceutically acceptable carrier and a plurality ofcryopreserved platelets having preserved platelet hemostatic activity.

In yet another embodiment, the pharmaceutical composition comprises aplurality of platelets, a plurality of a non-naturally occurringintracellular calcium chelator, a plurality of a non-naturally occurringsecond agent for inhibiting actin filament severing and apharmaceutically acceptable carrier. Exemplary non-naturally occurringcalcium chelators include the acetoxymethyl (AM) esters of the BAPTAfamily of calcium chelators (described below), and derivatives thereof.In a preferred embodiment, the calcium chelator is the acetoxymethylderivative of quin-2 and the agent for inhibiting actin polymerizationis cytochalasin B.

According to yet another aspect of the invention, a composition forpreventing cold-induced platelet activation is provided. The compositionincludes a plurality of a first agent for inhibiting actin filamentsevering and a plurality of a second agent for inhibiting actinpolymerization. The first and second agents are present in thecomposition in sufficient amounts so as to prevent cold-induced plateletactivation.

These and other aspects of the invention as well as various advantagesand utilities will be more apparent with reference to the detaileddescription of the preferred embodiments and in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates actin remodeling during plateletactivation;

FIG. 2 schematically illustrates actin remodeling during cold-inducedactivation of platelets;

FIG. 3 schematically illustrates actin remodeling during activation ofcalcium chelated platelets;

FIG. 4 graphically illustrates the time course of actin assembly incold-exposed or thrombin-treated platelets;

FIG. 5 graphically illustrates the prevention of cold-induced plateletactivation in platelets treated with quin-2AM and cytochalasin B;

FIG. 6 illustrates the morphology of resting human platelets at 37° C.;

FIG. 7 illustrates the morphology of human platelets exposed to 4° C.for 90 minutes;

FIG. 8 illustrates the morphology of human platelets treated with 40 μMquin-2AM and exposed to 4° C. for 90 minutes;

FIG. 9 illustrates the morphology of human platelets treated withquin-2AM and cytochalasin B and exposed to 4° C. for 90 minutes; and

FIG. 10 is a copy of an electron micrograph of a detergent-extractedcold-exposed platelet which was rapidly frozen, metal-shadowed andphotographed at about 40,000×magnification.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention embraces methods for preserving platelets withpreserved hemostatic activity, storage compositions for preventingcold-induced platelet activation and pharmaceutical compositions formediating hemostasis.

The compositions comprise a pharmaceutically acceptable carrier, aplurality of platelets, a plurality of a first agent for inhibitingactin filament severing and a plurality of a second agent for inhibitingactin polymerization. In a storage composition, the first and secondagents are present in the composition in sufficient amounts so as toprevent cold-induced platelet activation. As used herein, the phrase"cold-induced platelet activation" is a term having a particular meaningto one of ordinary skill in the art. Cold-induced platelet activation ismanifested by changes in platelet morphology, some of which are similarto the changes that result following platelet activation by, forexample, contact with glass. The structural changes indicative ofcold-induced platelet activation are most easily identified usingtechniques such as light or electron microscopy. On a molecular level,cold-induced platelet activation results in actin bundle formation and asubsequent increase in the concentration of intracellular calcium.Actin-bundle formation is detected using, for example, electronmicroscopy. An increase in intracellular calcium concentration isdetermined, for example, by employing fluorescent intracellular calciumchelators. Many of the above-described chelators for inhibiting actinfilament severing are also useful for determining the concentration ofintracellular calcium (Tsien, R., 1980, supra.). Cold-activatedplatelets also have a characteristically reduced hemostatic activity incomparison with platelets that have not been exposed to coldtemperatures. These differences in hemostatic activity are reflected indifferences in actin polymerization activity. Accordingly, varioustechniques are available to determine whether or not platelets haveexperienced cold-induced activation. Such techniques can be used toselect the concentrations of first and second agents that are necessaryto prevent cold-induced platelet activation.

The invention further embraces pharmaceutical compositions containingcryopreserved platelets that have preserved hemostatic activity.Hemostatic activity refers broadly to the ability of platelets tomediate bleeding cessation. Various assays are available for determiningplatelet hemostatic activity (Bennett, J. S. and Shattil, S. J., 1990,"Platelet function," Hematology, Williams, W. J., et al., Eds. McGrawHill, pp 1233-12250). However, demonstration of "hemostasis" or"hemostatic activity" ultimately requires a demonstration that plateletsinfused into a thrombocytopenic or thrombopathic (i.e., non-functionalplatelets) animal or human circulate and stop natural orexperimentally-induced bleeding.

Short of such a demonstration, laboratories use in vitro tests assurrogates for determining hemostatic activity. These tests, whichinclude assays of aggregation, secretion, platelet morphology andmetabolic changes, measure a wide variety of platelet functionalresponses to activation. There is, we believe, no in vitro method thatcan be directly translated into the in vivo setting. However, we believethat the tests disclosed herein are reasonably indicative of hemostaticfunction in vivo. Short of transfusion studies in animals and humans, wecan definitely state only that the methods disclosed herein prevent themorphological changes associated with cold-induced platelet activationand loss of in vitro responsiveness of platelets and that presumably,this translates into improved hemostasis in vivo. (See also Slichter, S.J., 1981 Vox Saug 40(Suppl 1):72-86).)

One indirect measure of hemostatic activity is the ability of plateletsto assemble actin monomers onto actin filaments. Freshly obtainedplatelets, which have not been subjected to cold temperatures, arehemostatically active and have substantial amounts of actinpolymerization activity. Platelets that have been subjected to coldtemperatures have increased basal polymerized actin, impaired survival,are less hemostatically active (Murphy, P. H. and Gardener F. H., 1969N. Engl. J. Med. 280:1094-1098; Handin, R. I. and Valeri, C. R., 1973 N.Engl. Med. 285:538-543) and have impaired actin polymerization activityin response to thrombin following rewarming (FIG. 5). In contrast, thecryopreserved platelets of the instant invention have an actinpolymerization activity that is greater than the actin polymerizationactivity of the cold-treated platelets. Thus "preserved hemostaticactivity" can be defined functionally (e.g., in terms of an actinpolymerization activity) to refer to an amount of hemostatic activitythat is greater than the hemostatic activity of a cold-treated platelet.In a preferred embodiment, the cryopreserved platelets have a hemostaticactivity (and corresponding actin polymerization activity) approachingthat of a platelet which has never been exposed to cold temperatures.Various assays are available for measuring actin polymerization andthereby obtaining a measure of platelet hemostatic activity (see e.g.,the pyrene-labeled rabbit skeletal muscle actin polymerization rateassay, Hartwig, J. and Janmey, P., 1989 Biochim. Biophys. Acta.3030:64-71).

As used herein, "actin filament severing" refers to the disruption ofthe non-covalent bonds between subunits comprising actin filaments.Actin filament severing in the platelet, presumably by gelsolin,requires an increase in the intracellular concentration of free calcium.Accordingly, in a preferred embodiment, the first agent for inhibitingactin filament severing is an intracellular calcium chelator. Exemplaryintracellular calcium chelators include the lipophillic esters (e.g.,acetoxymethyl esters) of the BAPTA family of calcium chelators, e.g.,QUIN, STIL, FURA, MAPTA, INDO, and derivatives thereof. See Cobbold andRink, 1987, supra. for a discussion of these intracellular chelators.

BAPTA is an acronym for 1,2-bis(2-aminophenoxy) ethaneN,N,-N',N'-tetraacetic acid. BAPTA and "BAPTA-like" compounds share ahigh selectivity for calcium over magnesium. As used herein,"BAPTA-like" refers to substituted derivatives of BAPTA andBAPTA-analogues which retain the essential calcium-chelatingcharacteristics of the parent (BAPTA) compound (see U.S. Pat. No.4,603,209, issued to Tsien, R., et al., the contents of which patent areincorporated herein by reference). By this definition, "BAPTA-like"compounds include compounds such as quin-1, quin-2, stil-1, stil-2,indo-1, fura-1, fura-2, fura-3, and derivatives thereof.

As used herein, quin-1 means2[[2-bis(carboxymethyl)amino]-5-methylphenoxy]methyl]-8-[bis(carboxylmethyl)amino]-quinoline.

As used herein, quin-2 means2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]-6-methoxy]-8-[bis(carboxymethyl)amino]quinoline.

As used herein, stil-1 means 1-(2-amino-5-[2-(4-carboxyphenyl)-E-ethenyl-1]phenoxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraaceticacid.

As used herein, stil-2 means1-(2(2-amino-5-[(2-(4-N,N-dimethylaminosulfonylphenyl)-E-ethenyl-1-]phenoxy)2-(2'-amino-5'methylphenoxy)ethane-N,N,N',N'-tetraaceticacid.

As used herein, indo-1 means1-(2-amino-5-[6-carboxyindolyl-2]1-phenoxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraaceticacid.

As used herein, fura-1 means1-(2-(4-carboxyphenyl)-6-amino-benzofuran-5-oxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraaceticacid.

As used herein, fura-2 means1-(2-(5'-carboxyoxazol-2'-yl)-6-aminobenzofuran-5-oxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraaceticacid.

As used herein, fura-3 means1-(2-(4-cyanophenyl)-6-aminobenzofuran-5-oxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraaceticacid.

The chemical structures for the above-identified calcium chelators areillustrated in U.S. Pat. No. 4,603,209, the contents of which patenthave been incorporated by reference.

As used herein, the phrase "pharmaceutically acceptable esters" (of theintracellular chelators) refers to lipophillic, readily hydrolyzableesters which are used in the pharmaceutical industry, especiallyalpha-acyloxyalkyl esters. See generally, references Ferres, H., 1980Chem. Ind. pp. 435-440, and Wermuth, C. G., 1980 Chem. Ind. pp. 433-435.

In a preferred embodiment, the intracellular chelator is theacetoxymethyl ester of quin-2 (Tsien, R., et al. (1982) J. Cell. Biol.94:325-334). Esterification transforms the hydrophilic chelator into alipophillic derivative that passively crosses the plasma membrane, andonce inside the cell, is cleaved to a cell-impermeant product byintracellular esterases. Preliminary biological tests of BAPTA and itslipophillic derivatives have so far revealed little or no binding tomembranes or toxic effects following intracellular microinjection(Tsien, R., supra.). Additional examples of intracellular calciumchelators are described in "Handbook of fluorescent Probes and ResearchChemicals," 5th edition, distributed by Molecular Probes, Inc., Eugene,Oreg.

We believe that addition of an intracellular calcium chelator mediatessevering by preventing activation of gelsolin. Accordingly, as usedherein, the phrase "agents for inhibiting actin filament severing" alsoembraces agents which directly inhibit gelsolin severing by affectingthe platelet polyphosphoinositides. Such agents include, for example,phosphotidylinositol 4-phosphate, phosphotidylinositol 4,5-bisphosphateand compounds structurally related thereto (Janmey, P. and Stossel, T.,1987 Nature 325:362-365; Janmey, P., et al., 1987 J. Biol. Chem.262:1228-12232).

The second agent (required for preventing cold-induced plateletactivation) inhibits barbed end actin polymerization. As used herein,"actin polymerization" refers to the process by which actin monomers("G-actin") are assembled onto the fast-growing ("barbed end") of actinfilaments ("F-actin"). Exemplary inhibitors of actin polymerizationinclude the class of fungal metabolites known as the cytochalasins andderivatives thereof (see e.g., "Biochemicals and Organic Compounds forResearch and Diagnostic Reagents" 1992, Sigma Chemical Company, St.Louis, Mo.).

Cytochalasin B is one of the best characterized of the cytochalasins. Inaddition to inhibiting actin polymerization, cytochalasin B enhances therate at which adenine nucleotides exchange on actin molecules and therate of ATP hydrolysis to ADP and orthophosphate. Cytochalasin B is alsoknown to inhibit the glucose transporter of eukaryotic cell membranes.The dihydro-derivatives of cytochalasins B and D inhibit actinpolymerization but do not exhibit this membrane-specific effect.

Despite the known interactions between cytochalasin B and biologicallyimportant proteins such as actin, few studies have been directed towardassessing toxicity of the cytochalasins. In vitro cell culture studieshave shown cytochalasin B to be non-cytotoxic at concentrations up to100 ug/ml (200 vM) for relatively short periods of time (about 2 hours)(Tsuyruo, et al., 1986 Biochem. Pharmacol. 35:1087-1090). Prolongedexposure of the cells in vitro results in reversible cytotoxicity withthe cytoxic effect eliminated upon removal of cytochalasin B from thecellular environment (Lipski, K., et al., 1987 Anal. Biochem.161:332-340).

Few studies have been conducted with the cytochalasins in vivo (see EPpatent publication number 0 297 946 A2, published 04.01.89). The tissuedistribution and toxicity (LD₅₀ =50 mg/kg) of cytochalasin B followingintraperitoneal administration to mice has been reported (Lipski, K., etal., supra.). Lipski et al. further report that cytochalasin Bdistributed rapidly into liver, renal fat, kidney, intestines mesentery,pancreas, spleen, and blood cells and was cleared from all but liverwithin 24 hours. However, only 35% of the injected cytochalasin B wasrecovered within a few minutes following injection, suggesting thatrapid oxidation of cytochalasin B to cytochalasin A, followed bysequestering of cytochalasin A in tissues, may account for the lowrecovery of cytochalasin B shortly after injection (Lipski, K., et al.,supra.). In contrast to cytochalasin B, dihydro-cytochalasin B is notsubject to oxidation to cytochalasin A. We believe that theconcentration of cytochalasin remaining in cryopreserved platelets willbe sufficiently low so that toxicity and/or sequestering of thecytochalasin will not be an issue. However, to avoid potentialsequestering of a cytochalasin oxidation product (e.g., cytochalasin A)in tissue, the dihydro-derivatives of the cytochalasins are employed ina preferred embodiment. In a most preferred embodiment, the second agentfor inhibiting actin polymerization is dihydro-cytochalasin B.

It is believed that the cytochalasins inhibit actin polymerization bycompeting with endogenous barbed end capping agents, e.g., gelsolin, andreducing the rate of monomer addition to the barbed end of growingfilaments. Based upon biochemical studies of the interactions betweengelsolin and actin in vitro (Examples 1 and 2), we believe that a classof membrane lipids, the polyphosphoinositides (ppIs), mediate thedissociation of gelsolin and related molecules from the barbed ends ofactin filaments. While much remains to be learned about these reactions,current information (Janmey, P. A., and Stossel, T., 1989 J. Biol. Chem.264:4825-4831) suggests that either biosynthesis or rearrangement ofppIs in response to platelet activating stimuli leads to aggregates ofthese lipids that induce the removal of gelsolin from the barbed ends ofactin filaments and the removal of certain actin monomer bindingproteins from actin subunits.

That the uncapping of gelsolin molecules from the barbed ends of actinfilaments (presumed to be mediated by ppIs) is separate from thecalcium-dependent severing step was demonstrated by contacting plateletswith quin-2AM prior to activation with thrombin (Example 2). FIG. 3schematically illustrates the actin remodeling events that occurredduring activation of the quin-2AM (calcium-chelated) platelets. Quin-2AMprevented the swelling and actin filament severing associated withplatelet activation as well as the subsequent extension of lamellipodialnetworks. Instead of collecting into filopodia, the actin bundlesappearing in activated Quin-2AM-treated platelets wound repeatedlyaround the interior of the platelet in dense coils, distorting grossplatelet morphology. Subsequent addition of calcium to the extracellularmedium resulted in fragmentation of the bundles and rounding of thedistorted platelets (Example 2). The latter result suggests that theaddition of extracellular calcium can supplement the extracellularcalcium stores to overcome intracellular calcium chelation. In view ofthese results, we believe that intracellular calcium chelation inhibitsactin filament severing by gelsolin but does not effect the ppI-mediateduncapping of core actin filaments or the desequestration (i.e.,dissociation of actin monomer binding proteins) of actin monomers andassembly of the monomers onto uncapped core filaments to generatefilopodial bundles. Since we believe that cold-induced changes in ppIsleads to the uncapping of gelsolin-blocked actin filaments, agents suchas peptides in the gelsolin sequence or peptides from a gelsolin-relatedprotein (e.g., villin) that bind to ppIs and inhibit gelsolin binding toppIs (Janmey P. A. et al., J. Biol. Chem. 267:11828-11838), alsotheoretically prevent cold-induced uncapping of filament by preventingactin filament severing. A patent application disclosing theabove-described peptides has been filed (U.S. application Ser. No.07/898607), the contents of which patent application are incorporatedherein by reference.

In a preferred embodiment, quin-2AM is the first agent for inhibitingactin filament severing and cytochalasin B or dihydro-cytochalasin B isthe second agent for inhibiting actin polymerization. As used herein,the "agents for inhibiting actin polymerization" include inhibitorshaving a similar mode of inhibition as the cytochalasins (presumablyppI-induced actin assembly), as well as inhibitors of actinpolymerization having alternative mechanisms.

Other xenobiotics having similar actions as the cytochalasins onplatelet actin assembly include the Coelenterate-derived alkaloids, thelatrunculins; the mushroom toxins, the virotoxins; and chaetoglobosinsfrom different fungal species. Additional agents known to inhibit actinpolymerization include actin monomer-binding proteins, profilin,thymosin, the vitamin D-binding protein (Gc globulin), DNAase I,actin-sequestering protein-56 (ASP-56), and the domain 1 fragments ofgelsolin and other actin filament-binding proteins (see e.g.,Kwiatkowski, O. J., et al., 1989 J. Cell Biol. 108:1717-1726; Way, etal. 1989 J. Cell Biol. 109:593-609; Hartwig, J. H. and Kwiatkowski, O.J., 1991 Curr. Opinion Cell Biol. 3:87-97; Vanderkerkhove, J. andVancompernolle, K., 1992 Curr. Opinion Cell Biol. 4:36-42). In addition,ADP-ribosylated actin reportedly acts like a barbed end-capping proteinand inhibits barbed end actin assembly (Aktories, K. and Wegner, A.,1989 J. Cell Biol. 109:1385. Accordingly, agents which ADP-ribosylateactin, e.g., certain bacterial toxins such as Clostridium botulinum C2and iota toxins, are embraced within the meaning of agents forinhibiting actin polymerization. Regardless of the mechanism ofinhibition, the actin polymerization inhibitors have in common theability to penetrate the plasma membrane.

The inhibitor of actin polymerization, may be contacted with theplatelets at any time prior to subjecting the platelets to thecryopreservation temperature. Accordingly, the second agent may becontacted with the platelets at the same time as the first agent isadded or before or after addition of the first agent. In a preferredembodiment, the first agent is contacted with the platelets beforecontacting the second agent with the platelets.

The agents are added to platelets that are kept between about roomtemperature and 37° C. Following treatment, the platelets are cooled toabout 4° C. In a preferred embodiment, the platelets are collected intoa platelet pack or bag according to standard methods known to one ofskill in the art. Typically, blood from a donor is drawn into a primarybag which may be joined to at least one satellite bag, all of which bagsare connected and sterilized before use. In a preferred embodiment, theplatelets are concentrated (e.g. by centrifugation) and the plasma andred blood cells are drawn off into separete satellite bags (to avoidmodification of these clinically valuable fractions) prior tosequentially adding the first and second agents. Platelet concentrationprior to treatment also minimizes the amounts of first and second agentsrequired for cryopreservation, thereby minimizing the amounts of theseagents that are eventually infused into the patient.

In a most preferred embodiment, the first and second agents arecontacted with the platelets in a closed system, e.g. a sterile, sealedplatelet pack, so as to avoid microbial contamination. Typically, avenipuncture conduit is the only opening in the pack during plateletprocurement or transfusion. Accordingly, to maintain a closed systemduring treatment of the platelets with the first and second agents, theagents are placed in a relatively small, sterile container which isattached to the platelet pack by a sterile connection tube (see e.g.,U.S. Pat. No. 4,412,835, the contents of which are incorporated hereinby reference). The connection tube is reversibly sealed according tomethods known to those of skill in the art. After the platelets areconcentrated, e.g. by allowing the platelets to settle and squeezing theplasma out of the primary pack and into a satellite bag according tostandard practice, the seal to the container(s) including the first andsecond agents is opened and the agents are introduced into the plateletpack. In a preferred embodiment, the first and second agents arecontained in separate containers having separate resealable connectiontubes to permit the sequential addition of first and second agents tothe platelet concentrate.

Following contact with the first and second agents, the treatedplatelets are stored at a cryopreservation temperature. As used herein,"cryopreservation temperature" refers to a temperature that is less thanstandard platelet storage temperatures, e.g., less than about 22° C. Ina preferred embodiment, the cryopreservation temperature ranges fromabout 0° C. to about 4° C. In contrast to platelets stored at, forexample, 22° C., platelets stored at cryopreservation temperatures havesubstantially reduced metabolic activity. Thus, platelets stored at 4°C. are metabolically less active and therefore do not generate largeamounts of CO₂ compared with platelets stored at, for example, 22° C.(Slichter, S., 1981, supra.). Dissolution of CO₂ in the platelet matrixresults in a reduction in pH and a concommittant reduction in plateletviability (Slichter, S., 1981, supra.). Accordingly, conventionalplatelet packs are formed of materials that are designed and constructedof a sufficiently permeable material to maximize gas transport into andout of the pack (O₂ in and CO₂ out). The prior art limitations inplatelet pack design and construction are obviated by the instantinvention, which permits storage of platelets at cryopreservationtemperatures, thereby substantially reducing platelet metabolism anddiminishing the amount of CO₂ generated by the platelets during storage.

According to another aspect of the invention, a method for making apharmaceutical preparation for administration to a mammal is provided.The method comprises preparing the above-described cryopreservedplatelet preparation, warming the platelet preparation, neutralizing thefirst and second agents and placing the neutralized platelet preparationin a pharmaceutically acceptable carrier. In a preferred embodiment, thecryopreserved platelets are warmed to room temperature (about 22° C.)prior to neutralization. In a most preferred embodiment, the plateletsare contained in a pharmaceutically acceptable carrier prior to contactwith the first and second agents and it is not necessary to place theplatelet preparation in a pharmaceutically acceptable carrier followingneutralization.

As used herein, the terms "neutralize" or "neutralization" refer to aprocess by which the first and second agents are rendered substantiallyincapable of further action in the preparation as agents for inhibitingactin filament severing and inhibiting actin polymerization,respectively. In a preferred embodiment, the cryopreserved platelets areneutralized by dilution, e.g., with a suspension of red blood cells.Alternatively, the treated platelets can be infused into the recipient,which is equivalent to dilution in millimolar calcium and into a redblood cell suspension. This method of neutralization advantageouslymaintains a closed system and minimizes damage to the platelets.

An alternative method to reduce toxicity is by inserting a filter in theinfusion line, the filter containing, e.g. activated charcoal or animmobilized anti-cytochalasin antibody, to remove the first and secondagents. Either or both of the first and second agents also may beremoved or substantially diluted by washing the treated platelets. Ininstances in which the first agent is an intracellular calcium chelator,the first agent is preferably neutralized by the addition of unchelatedcalcium to the cryopreserved platelet preparation. The unchelatedcalcium is added to the preparation at a concentration in excess of theintracellular calcium chelator concentration.

The invention further provides a method for mediating hemostasis in amammal. The method includes administering the above-describedpharmaceutical preparation to the mammal. Administration of thecryopreserved platelets may be in accordance with standard methods knownin the art. According to one embodiment, a human patient is transfusedwith red blood cells before, after or during administration of thecryopreserved platelets. The red blood cell transfusion serves to dilutethe administered, cryopreserved platelets, thereby neutralizing thefirst and second agents.

Also within the scope of the invention are storage compositions andpharmaceutical compositions for mediating hemostasis.

In one embodiment, the compositions comprise apharmaceutically-acceptable carrier, a plurality of platelets, aplurality of a first agent for inhibiting actin filament severing and aplurality of a second agent for inhibiting actin polymerization. Thefirst and second agents are present in the composition in sufficientamounts so as to prevent cold-induced platelet activation.

The criteria for selecting the amounts of first and second agents forpreventing cold-induced platelet activation are: (1) the first agentmust be present in the composition in an amount which inhibits actinfilament severing and (2) the second agent must be present in thecomposition in an amount that inhibits actin filament polymerization.Preferably, they are present in amounts whereby after cryopreservationand neutralization, the platelets have preserved hemostatic activity.The amounts of first and second agents which prevent cold-inducedplatelet activation can be selected by exposing a preparation ofplatelets to increasing amounts of these agents, exposing the treatedplatelets to a cryopreservation temperature and determining (e.g., bymicroscopy) whether or not cold-induced platelet activation hasoccurred. Alternatively, the amounts of first and second agents can bedetermined functionally by exposing the platelets to varying amounts offirst and second agents, cooling the platelets as described herein,warming the treated (chilled) platelets, neutralizing the platelets andtesting the platelets in a hemostatic activity assay to determinewhether the treated platelets have preserved hemostatic activity.

For example, to determine the optimal concentrations and conditions forpreventing cold-induced activation by a first agent that is anintracellular calcium chelator and by a second agent that is acytochalasin, increasing amounts of these agents are contacted with theplatelets prior to exposing the platelets to a cryopreservationtemperature. The optimal concentrations of the first and second agentsare the minimal effective concentrations that preserve intact plateletfunction as determined by in vitro tests (e.g., observing morphologicalchanges in response to glass, thrombin, cryopreservation temperatures;ADP-induced aggregation; actin polymerization) followed by in vivo testsindicative of hemostatic function (e.g., recovery, survival andshortening of bleeding time in a thrombocytopenic animal or recovery andsurvival of ⁵¹ Cr-labeled platelets in human subjects).

New compounds also can be screened for their ability to act as first andsecond agents in preventing cold-induced platelet activation. Theamounts of previously untested first and second agents necessary toprevent cold-induced platelet activation can be determined by selectingan amount of first agent which inhibits actin filament severing and byselecting an amount of second agent which inhibits actin polymerization.As previously noted, the severing of actin filaments is detectable byelectron microscopy or other published procedures (see e.g. Janmey, P.A. and Stossel, T. P., 1987 Nature 325:362-365). One method forselecting an amount of an untested first agent for inhibiting actinsevering is by treating intact platelet preparations (containingincreasing amounts of the first agent) with cytochalasin B (to inhibitbarbed end polymerization activity), activating thepolymerization-inhibited platelets (e.g., by exposure to a cryogenictemperature) and observing (by microscopy) structural changes in thepolymerization-inhibited, activated platelets. Thepolymerization-inhibited, activated control platelets (i.e., plateletsthat were not exposed to first agent) exhibit actin filament severing(as observed by electron microscopy) but do not extend the lamellipodiaor filopodia characteristic of actin polymerization because actinpolymerization is inhibited. The polymerization-inhibited, activatedplatelets (exposed to increasing amounts of the first agent) exhibitdecreasing amounts of actin severing (as observed by electronmicroscopy). According to this method, the amount of first agent whichis necessary to prevent cold-induced platelet activation is that amountwhich empirically inhibits actin severing. For first agents that areintracellular calcium chelators, the amount of the first agent thatinhibits actin severing is, in part, dependent upon the affinity andspecificity of the intracellular chelator for calcium. In a preferredembodiment, the first agent is Quin-2AM which is present in thecomposition at a concentration of about 10⁻¹⁶ mole/platelet.

Similarly, various assays are available for selecting an amount ofsecond agent that inhibits actin monomer assembly onto actin filaments(actin polymerization). For example, a pyrene-labeled actinpolymerization assay has previously been described (Hartwig, J. andJanmey, P. 1989 Biochim. Biophys. Acta 1010:64-71). Pyrene actinassembly onto actin filaments is completely inhibited at the barbed endby 2 μM cytochalasin B (Examples 1 and 2). Thus, cytochalasin Binhibitable activity in the pyrene-labeled polymerization assay isdefined as "barbed end" actin assembly (polymerization). Accordingly,the amount of an untested second agent for inhibiting actinpolymerization is determined, for example, by substituting the untestedsecond agent for cytochalasin B in the pyrene labeled polymerizationassay (using increasing amounts of the untested second agent) andselecting the concentration of second agent that inhibits actin assemblyonto the barbed end of actin filaments. In a preferred embodiment, thesecond agent is cytochalasin B which is present in the composition at aconcentration of about 10⁻¹⁸ mole/platelet. In a most preferredembodiment, the second agent is dihydro-cytochalasin B which is presentin the composition at a concentration of about 10⁻¹⁸ to about 10⁻¹⁷mole/platelet.

In yet another embodiment, the pharmaceutical composition comprises aplurality of platelets, a plurality of a non-naturally occurringintracellular calcium chelator, a plurality of a non-naturally occurringsecond agent for inhibiting actin polymerization and a pharmaceuticallyacceptable carrier. As used herein, the term "non-naturally occurring"refers to a molecule which is not present in platelets as they exist incirculating blood. Exemplary non-naturally occurring intracellularcalcium chelators are the above-described lipophilic derivatives of theBAPTA family of calcium chelators. Exemplary non-naturally occurringsecond agents for inhibiting actin polymerization include theabove-described cytochalasins and derivatives thereof, as well asfragments of larger molecules which are present in platelets as theyexist in circulating blood.

According to yet another aspect of the invention, a composition foraddition to platelets to prevent cold-induced platelet activation isprovided. The composition includes a plurality of a first agent forinhibiting actin filament severing and a plurality of a second agent forinhibiting actin polymerization. The first and second agents are presentin the composition in amounts that prevent cold-induced plateletactivation. In a preferred embodiment, the first agent is theacetoxymethyl ester of quin-2 (quin-2AM) and the second agent iscytochalasin B.

EXAMPLES

The instant invention provides methods and pharmaceutical compositionsfor the cryopreservation of platelets with preserved hemostaticactivity. The following examples illustrate representative utilities ofthe instant invention.

Materials and Methods:

A. Preparation of Resting Platelets

Human blood from healthy volunteers, drawn into 0.1 vol of Aster-Jandlanticoagulant, was centrifuged at 110 g for 10 min. The platelet-richplasma was gel-filtered through a Sepharose 2B column equilibrated andeluted with a solution containing 145 mM NaCl, 10 mM Hepes, 10 mMglucose, 0.5 mM Na₂ HPO₄, 5 mM KCl, 2 mM MgCl₂, and 0.3% BSA, pH 7.4(platelet buffer). 2 U/ml apyrase was added to the platelet suspensionand the cells were left standing for 60 min. at 37° C. as previouslyreported (Hartwig, J., and M. DeSisto, 1991 J. Cell Biol. 112:407-425;Hartwig, J., et al. 1989 J. Cell Biol. 109:1571-1579). To maintaincytosolic calcium at or below its resting level during cell activation,cells were loaded with 30 uM Quin-2AM during minutes 30-60 of the restperiod. The effect of Quin-2 was reversed by the addition of 1 mM CaCl₂to the bathing media before centrifugation of the Quin 2-loaded cellsonto the coverslips or after the cells had been attached and formedfilopodia on the coverslip. Glass-adherent, Quin-2 loaded cells werealso treated with 1 mM CaCL₂ and 20 nM of the ionophore A23187 for 15seconds (s) and then detergent permeabilized. In some cases, plateletswere used directly from platelet-rich plasma by diluting it 1:20 withplatelet buffer containing, in addition, 0.1 mM EGTA and 2 U/ml apyrase.The diluted cells were incubated for 30 min. at 37° C. to insure aresting state.

B. Activation of Platelets

Platelet suspensions were activated by the addition of 1 U/ml ofthrombin (hereafter called thrombin-activated) for 15-30 seconds instudies of nucleation activity. Activation was terminated bypermeabilizing the cells as detailed below. Glass activation was usedfor the morphological studies. Cells were glass-activated bycentrifugation onto polylysine-coated glass coverslips at 250 g for 5min. Coverslips were placed in the bottom of multiwell plates (24 or 96wells), covered with 0.25 ml of platelet suspension, and centrifuged at37° C. in a Sorvall HB-6000 centrifuge using multiwell carriers.

C. Fluorescence Measurement of Actin Assembly in Lysates from Restingand Activated Cells

The effect of cell lysates on the rate and extent of pyrene-labeledrabbit skeletal muscle actin was determined as described previously(Hartwig, J., and P. Janmey, 1989 Biochim. Biophys. Acta. 1010:64-71).Suspensions of resting or thrombin-activated cells at concentrations of1.4×10⁸ /ml were permeabilized by the addition of 0.1 volume of 60 mMPipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl₂, 0.75% Triton and 42 nMleupeptin, 10 mM benzamidine, and 0.123 mM aprotinin to inhibitproteases (Schliwa, J., et al., 1981 Proc. Natl. Acad. Sci. USA80:5417-5420). 100 ul of detergent lysate was added to 190 ul of 100 mMKCl, 2 mM MgCl₂, 0.5 mM ATP, 0.1 mM EGTA, 0.5 mM dithiothreitol, and 10mM Tris, pH 7.0 and the polymerization rate assay was started by theaddition of monomeric pyrene-labeled rabbit skeletal muscle actin to afinal concentration of 2 uM. The relative contribution of nuclei withbarbed or pointed ends in the cell lysates was determined by adding 2 uMcytochalasin B to the pyrene nucleation assay system. Pyrene actinassembly onto actin filament nuclei has been shown to be completelyinhibited at the barbed end by 2 uM cytochalasin B. Cytochalasin Binhibitable activity in the nucleation assay is, therefore, defined as"barbed" end assembly. Activity not inhibited by cytochalasin B isconsidered pointed end assembly. The stability of nucleation activity incell lysates was tested by comparing the stimulatory effect of freshlysate on actin assembly with lysates allowed to stand for 30 s to 30min at 37° C. before addition to the assembly assay. To determine if themeasured stimulation of actin assembly and its decay with time was dueto the growth of pyrene-actin addition onto cellular filaments subjectto depolymerization in the diluted lysate, 0.1 uM phalloidin orphallacidin was added to the cell lysates during their preparation tostabilize the filaments. As shown in the results, all nucleationactivity present in resting and activated cells was associated with thedetergent-insoluble cytoskeleton. However, we also determined that thesoluble phase from cells permeabilized with detergent in the presence ofEGTA contained calcium dependent nucleation activity. Detergent lysatesfrom resting and thrombin-activated cells (30 s, 1 U/ml) werecentrifuged at 10,000 g for 2 min at room temperature in amicrocentrifuge. The supernatant was removed and added to thepyrene-based nucleation assay in the presence of 1 mM EGTA or CaCl₂. Theamount of pointed end activity in these soluble extracts was determinedby adding a final concentration of 2 uM cytochalasin B to thepyrene-actin assembly assay.

The effect of inhibiting barbed end actin assembly in thrombin-activatedcells before detergent lysis on the amount of nucleation activity wasdetermined by preincubating resting platelet suspensions with 2 uMcytochalasin B for 5 min. Because it was necessary to wash out thecytochalasin B from some of the cytoskeletons before addition of thecell lysates to the pyrene assembly assay, the cells were first attachedto glass coverslips while still in the presence of cytochalasin B. Thiswas accomplished by sedimenting 0.3 ml of cell suspension onto a 12 mmround glass coverslip for 5 min at 250 g. Individual coverslips wereremoved, treated with thrombin for 15 s in the presence of cytochalasinB, permeabilized with 1×PHEM-Triton buffer containing 2 uM cytochalasinB for 15 s and then washed in PHEM buffer in the presence or absence ofcytochalasin B. Coverslips were then immediately assayed for theirability to promote actin filament assembly as previously described by ususing glass adherent macrophage cytoskeletons (Hartwig, J., and P.Janmey, 1989 Biochim. Biophys. Acta. 1010:64-71).

D. Morphological Studies

Light microscopy and electron microscopy of platelets and cytoskeletonswere performed according to standard methods, see e.g., Hartwig, J.,1992, J. Cell Biol. 118(6):1421-1442. The localization of gelsolin inplatelets was performed by gold labeling of cytoskeletons from restingand activated cells with antibodies to gelsolin. The affinity-purifiedgoat anti-rabbit macrophage gelsolin IgG was described earlier (Hartwig,J., and M. DeSisto, 1991 J. Cell Biol. 112:407-425; Hartwig, J., and P.Shevlin, 1986 J. Cell Biol. 103:1007-1020).

Example 1 Actin Nucleation Activity in Resting and Activated Cells

To understand how new filament assembly (polymerization) is initiatedduring cell activation, the nature and amount of nucleation activity indetergent lysates from resting and thrombin-activated cells wascharacterized using a pyrene-actin assembly system in vitro. As shown inTable 1, lysates of resting cells permeabilized with Triton X-100 hadonly a small stimulatory effect on the rate at which pyrene-actinassembled in solutions containing 0.1M KCl and 2 mM MgCl₂. This smallincrease in the rate of actin assembly by resting lysates was probablydue primarily to the addition of pyrene monomers onto theslow-exchanging filament ends (pointed end of S1-labeled fibers) becauseaddition of 2 uM cytochalasin B to lysates from resting cells, whichblocks exchange at the high affinity ("barbed") ends, had only a smalleffect on the amount of nucleation activity measured in the pyreneassay. From the kinetics and extent of pyrene assembly and rates ofaddition of monomers to the filament ends, 2,000 pointed filament endsare present in a resting cell. Lysates from cells activated with 1 U/mlof thrombin for 15-30 s before permeabilization, however, increased thepyrene-actin assembly rate three- to four-fold relative to restinglysates (Table 1). In contrast to the resting lysates, the stimulatoryeffect in these lysates was abolished by the addition of 2 uMcytochalasin B to the actin assembly assay (Table 1). Since cytochalasinB blocks new actin assembly in both intact, thrombin-activated platelets(Casella, J., et al., 1981 Nature (Lond.) 293:302-305; Fox, J. and D.Phillips, 1981 Nature (Lond.) 292:650-652) and in lysates fromthrombin-activated cells, filament assembly in platelets must occurpredominantly on the fast growing (barbed) end of filaments. Dependingon the experiment, 410-570 barbed ends would have been required onaverage in each platelet to increase the rate of actin assembly by thedetermined extents (Table 1).

Before centrifugation, lysates increased the actin assembly rate by1.528±0.11 nM s⁻¹ (mean ±SD) relative to actin alone. Centrifugation oflysates at 10,000 g for 2 min, which sediments aggregates of cellularactin fibers but not individual actin filaments, removed 99% of thenucleation activity induced by thrombin. This result indicates that allbarbed end nucleation activity measured in EGTA-containing buffer wasassociated with the low-speed sedimentable platelet cytoskeleton.

In all of the nucleation experiments described above, the calcium ionconcentration was low because all solutions contained EGTA to chelatecalcium. Although raising the calcium concentration of assay solutionsinto the micromolar range by addition of sufficient CaCl₂ had no effecton the barbed end nucleation activity that was sedimentable in lysatesof thrombin-activated platelets, the soluble fraction remaining afterremoval of cytoskeletons also contained a large amount ofcalcium-dependent actin nucleation activity (Table II). This activitywas, however, in contrast to the calcium-insensitive sedimentableactivity, completely unaffected by 2 uM cytochalasin B, demonstratingthat it promotes actin assembly only in the pointed (non-barbed end)direction. No calcium-activated soluble nucleation activity wasdetectable in lysates from resting cells.

                                      TABLE I                                     __________________________________________________________________________    Calcium-insensitive Cytoskeletal Nucleation Activity                                Assembly rate                                                                         Assembly rate                                                                         Subunits added to                                                                      Subunits added to                                    (Pointed end)                                                                         (barbed end*)                                                                         pointed ends**                                                                         barbed ends***                                                                         Pointed Barbed                        Treatment                                                                           nM s.sup.-1                                                                           nM s.sup.-1                                                                           x10.sup.10 s.sup.-1                                                                    x10.sup.10 s.sup.-1                                                                    nuclei/platelet                                                                       nuclei/platelet               __________________________________________________________________________    Resting                                                                             0.21 ± 0.04                                                                        0.04 ± 0.02                                                                        3.7 ± 0.70                                                                           0.7     2,000 ± 380                                                                         50 ± 25                   Activated                                                                           0.24 ± 0.02                                                                        0.58 ± 0.03                                                                        4.3 ± 0.35                                                                          10.4     2,500 ± 200                                                                        410 ± 15                   __________________________________________________________________________     *The barbed end assembly rate is calculated by subtracting the assembly       rate in the presence of 2 μM cytochalasin B from the assembly rate in      the absence of cytochalasin B.                                                **Initial pointed end addition rate in 2 μM actin solution was 1.2         subunits s.sup.-1.                                                            ***Initial barbed end addition rate in 2 μM actin solution was 18          subunits s.sup.-1.                                                       

In reference to Table I, gel-filtered platelets were suspended to aconcentration of 1.4×10⁸ /ml in platelet buffer, rested for 30 minutesat 37° C., and then treated with or without 1 U/ml of thrombin for 15 s.The cells were permeabilized by the addition of 0.1 vol of 10× PHEMbuffer (Schliwa, M., et al., 1981 Proc. Natl. Acad. Sci. USA80:5417-5420) containing 0.75% Triton X-100 and protease inhibitors. 110μl of platelet lysate was added to 180 μl of 0.1M KCl, 0.5 mM ATP, 2 mMMgCl₂, 0.3 mM beta-mercaptoethanol, and 2 mM Tris, pH 7.0. The rateassay was started by the addition of pyrene-labeled actin monomers to afinal concentration of 2 μM. The final volume was 0.3 ml. 2 μMcytochalasin B was added to determine the amount of total activityrelated to the barbed filament end. There were 1.4× 10⁷ platelets perassay. Data are expressed as mean ±SD, n=4.

                  TABLE II                                                        ______________________________________                                        Calcium-sensitive Soluble Nucleation Activity                                                            Subunits                                                                      added to                                                            Assembly  pointed Pointed                                             2 μM Rate*     ends**  nuclei/                                    Treatment                                                                              CB      nM s.sup.-1                                                                             x10.sup.10 s.sup.-1                                                                   platelet                                   ______________________________________                                        Resting  +       ND        --      --                                         Resting          ND        --      --                                         Activated                                                                              +       0.55 ± 0.02                                                                           1.0 ± 0.05                                                                        5,000 ± 250                             Activated        0.54 ± 0.03                                                                          0.97 ± 0.05                                                                        4,850 ± 243                             ______________________________________                                    

In reference to Table II, lysates were prepared from restingthrombin-treated cells with detergent and centrifuged in a SorvallMicrospin 12S at 13,000 rpm for 1 min. Nucleation activity remaining inthe supernatant after removal of the cytoskeletal fraction wasdetermined. Centrifugation of lysates removed allcytoskeletal-associated nucleation activity from the resultantsupernatants. There were 2.0×10 platelets per assay. The data areexpressed as mean ±SD, n=4. NC=not detectable. The barbed end assemblyrate, initial pointed end addition rate and initial barbed end additionrate are as described for Table I.

Example 2 Evidence for the Role of Calcium in CytoskeletalRearrangements Occurring with Platelet Activation

A. Quin-2-loaded Platelets Attach to Glass Extend Filopodia, but Do NotSpread

Loading platelets with 30 uM Quin-2AM had no effect on the structure ofthe resting cells observed in the light microscope or cytoskeletonsprepared from these resting cells (data not shown). However, themorphologies of glass-activated cells differed from untreated cellsspreading on coverslips. As illustrated schematically in FIG. 3,platelets loaded with Quin-2 and then glass activated extend filopodiabut not lamellipodia. Filopodia were 2-5 um in length, thicker indiameter relative to those from control cells, and had bulbous endings(FIG. 3). Although filopodia extended from these cells, the cell shaperemained discoid with dimensions near those of the resting cell. In theelectron microscope, the surface of intact cells retained the pits ofthe open canalicular system (OCS). While most of the cells hadfilopodia, some other morphologies were also apparent. Mostglass-activated Quin-2-loaded platelets extended one prominent filopod,but a few simply elongated or made spherical protrusions at theirmargins. As detailed below, these different morphologies resulted fromrelated cytoskeletal actin rearrangements. If Quin-2-loaded cells werebathed in medium containing millimolar calcium, normal spreading ofcells resulted on the glass surfaces (i.e., cells spread bothlamellipodia and filopodia.

When observed in the electron microscope, cytoskeletons prepared fromQuin-2-loaded and glass-activated cells lack lamellipodial networks attheir margins. Instead, these cytoskeletons are composed exclusively oflong filaments running parallel to the cell margin. These filamentsappear to derive from filaments originating in the cell center whichturn and run along the cytoskeletal edges. Filopodia in cytoskeletonsfrom Quin-2-loaded cells are filled with actin filaments originatingnear the middle of the cytoskeleton, but these filaments do not end nearthe tips of the filopodia as in control cells. Instead, these filamentsmake U-turns and run back into the body of the cytoskeleton. Thesefilament loops, therefore, appear to produce the bulbous enlargements atthe ends of filopodia in these cells. Not all actin fibers enteringfilopods make U-turns. A few of the filaments within filopodia end neartheir tips. Examination of cytoskeletons from cells displaying simply anelongated shape without a filopod reveals them to have internal bundlesof filaments. Fibers coming off the ends of these bundles turn and runin parallel with filaments in the cytoskeletal margins or turn and runback toward the middle of the cytoskeletons instead of exiting to formfilopodia. Cytoskeletons of bleb forms also share these features. Blebsat the cytoskeletal edges are composed of loops of actin filaments withsome underlying straight filaments.

B. Quin-2 Loading Diminishes Thrombin-stimulated Nucleation Activity

Nucleation activity in lysates from Quin-2-loaded and control cells wascompared after thrombin activation. Lysates from activated cells loadedwith Quin-2 had only 28% of the nucleation activity of lysates fromuntreated and activated cells when assayed immediately. When directlycompared, the total number of nuclei was equivalent to that remaining incontrol lysates incubated for 2 min. before addition to the assemblyassay. Although the total nucleation activity was reduced, its stabilitywas increased in lysates of Quin-2-chelated, thrombin-activated cells.Nucleation activity in the detergent lysates from Quin-2-loaded cellswas more stable, and no loss in its activity occurred in lysatesincubated for as long as 10 min. before addition to the assembly assay.In contrast to lysates from unchelated cells, phalloidin had no effecton the nucleation activity in lysates from Quin-2-loaded cells. Thestability of actin filament nuclei in the Quin-2-loaded cells couldresult either from their being considerably longer in length comparedwith control cells or from their being coated with proteins that retarddepolymerization. The former alternative finds support in the electronmicroscope where the periphery of Quin-2-loaded and activated cells werereplete with long fibers. Filaments of lengths greater than or equal to1.5 um would have survived to nucleate in the assay.

The above experiments have shown that long actin filaments preexistingin resting platelets shorted in a calcium-dependent fashion during cellactivation with thrombin or glass stimuli and that these short filamentsthen become templates for the assembly of lamellipodial networks. Twopossibilities exist for the formation of this short filament populationduring cell activation. Filaments forming the resting cytoskeleton couldbe fragmented into smaller pieces or the resting actin cytoskeletoncould disassemble to monomers and be replaced by a new population ofshort filaments. The following experiments address the mechanism of thisshort filament formation.

C. Readdition of Calcium to Quin-2-loaded Cells Rapidly Dissolved theActin Filament Bundles in These Cells

Filopodial forms generated by Quin-2-loading and glass activation ofplatelets were rapidly converted to forms resembling activated,unchelated platelets when the buffer bathing the cells was replaced withone containing millimolar calcium. Bundles were rapidly reorganized intolamellipodial networks. The effect of added external calcium in thepresence of the ionophore A23187 was more dramatic. Within seconds, thecytoskeletons of previously chelated cells that contained large actinfilament bundles were completely disrupted, leaving a fibrous residuelacking actin filaments. This disruption occurred too rapidly to beexplained by filaments depolymerizing from their ends. Many actinfilaments were also scattered over the surface of the coverslip.

D. Cytochalasin B Does Not Affect the Amount of Nucleation Activity inLysates of Activated Cells

In the experiments described above, cytochalasin B added to lysatesserved as a test for the direction (barbed versus pointed) of actinassembly off of nuclei present. In the following experiments,cytochalasin B incubated with intact platelets and later diluted toconcentrations in lysates below which it blocks actin assembly permittedthe assessment of morphological changes and determination of whetheractin nucleation activity appears after platelet activation under acondition in which the bulk of cytoplasmic actin cannot assemble.

The generation of nucleation activity after thrombin treatment in thepresence of cytochalasin B was demonstrated using the pyrene-labeledactin assembly assay. As shown in Table III, cytoskeletons fromthrombin-activated and cytochalasin B-treated cells stimulate the rateof pyrene-actin assembly in vitro four-fold compared with resting cellsincubated with cytochalasin B in parallel (to levels comparable tolysates from thrombin-activated cells) when cytochalasin was presentwhile the cells were undergoing activation but washed away beforedetermining nucleation activity. Cytochalasin B treatment of restingcells did not by itself result in nucleation activity. In addition, therate of actin assembly from the barbed filament ends was near that incells not exposed to cytochalasin B (compared with Table I). Therefore,these experiments demonstrate that the short filaments found incytoskeletons from activated cells do not derive from the de novoassembly of actin monomers onto some unspecified barbed end nucleatingagent, because cytochalasin B did not inhibit their formation.

To demonstrate that the short actin filaments formed in cells activatedin the presence of cytochalasin were calcium dependent, the normal risein cytosolic calcium was inhibited by loading these cells with Quin-2and then attaching them to glass by centrifugation in the presence ofcytochalasin B. The morphology of these cells was unchanged from that ofresting cells and a cytoskeleton prepared from these cells resembles thestructure of the resting platelet cytoskeleton. The cytoskeleton of suchcells was discoid and covered with its dense membrane skeleton.

                                      TABLE III                                   __________________________________________________________________________    Effect of Cytochalasin B on the Nucleation                                    Activity in Activated Cytoskeletons                                                 Assembly rate                                                                         Assembly rate                                                                         Subunits added to                                                                      Subunits added to                                    (pointed end)                                                                         (barbed end*)                                                                         pointed ends**                                                                         barbed ends***                                                                         Pointed Barbed                        Treatment                                                                           nM s.sup.-1                                                                           nM s.sup.-1                                                                           x10.sup.10 s.sup.-1                                                                    x10.sup.10 s.sup.-1                                                                    nuclei/platelet                                                                       nuclei/platelet               __________________________________________________________________________    Resting                                                                             0.55 ± 0.15                                                                        0.18 ± 0.23                                                                        9.95 ± 3.30                                                                          2.0     1,980 ± 660                                                                         50 + 20                      Activated                                                                           0.55 + 0.18                                                                           1.59 ± 0.30                                                                        9.95 ± 3.30                                                                         39.0     1,980 ± 660                                                                        380 + 50                      __________________________________________________________________________

In reference to Table III, resting platelets were incubated with 2 μMcytochalasin B for 5 min, then adhered by centrifugation to 12-mm roundglass coverslips coated with polylysine. Cells on the coverslips wereexposed to 1 U/ml of thrombin for 30 s in the presence of cytochalasin Band then permeabilized with PHEM-Triton buffer. Some coverslips werewashed rapidly (1-2×) through PHEM that did not contain cytochalasin toremove this agent and added to the pyrene-labeled actin assembly system.Cytoskeletons from thrombin-treated cells markedly stimulated the rateof actin assembly nucleation activity upon the removal of thecytochalasin B. Cytochalasin B treatment of resting cells did notincrease the amount of nucleation activity in resting cytoskeletons.There were 4.2×10⁷ platelets per assay. The data are expressed as mean±SD, n=4. The barbed end assembly rate, initial pointed end additionrate and initial barbed end addition rate were as described in Table I.

E. Location of Gelsolin in Resting and Activated Cytoskeletons

The results of the experiments described above implicatecalcium-activated actin filament severing as an important step in theremodeling of the resting cytoskeleton into the activated form. Gelsolinaccounts for 0.5% of platelet total protein, yielding a molar ratio ofgelsolin to actin of about 1:80, and is an excellent candidate to causethe actin severing observed during platelet activation. Gelsolin waslocalized in resting and activated cytoskeletons by immunoelectronmicroscopy. The cytoskeletal gelsolin identified with anti-gelsolin IgGand colloidal-gold particles were found in clusters bound near themembrane skeleton-actin filament interface in thin sections. Todetermine whether this gelsolin was associated with the ends of actinfilaments at this interface or linked to the membrane skeleton, it waslocalized in mechanically opened cytoskeletons (Hartwig, J. and DeSisto,M., 1991 J. Cell Biol. 112:407-425) from resting cells. Micrographs ofthese preparations demonstrated that: (a) gelsolin does not associatewith the membrane skeleton per se; (b) gelsolin does associate with theactin filament core lining its surface; (c) gold particles are clusteredin the core; and (d) is on the ends of at least some of the 10-nmfilaments knocked out of the cytoskeletons by the mechanical treatmentand is associated with filaments within the filamentous core to themembrane skeleton. Since the bulk of gelsolin released by detergenttreatment of resting cells is gelsolin free (>95%), the large number ofgold particles not bound to actin filament ends in these specimens isexpected.

As the cytoskeleton changes during spreading, gelsolin also changes inits distribution from the resting condition. Gelsolin-reactive goldparticles located in the lamellipodial zone of the cytoskeleton.Labeling occurred preferentially on one filament end, and less oftenalong filaments. The filament ends decorated with gelsolin-gold werefree, were attached to the substratum or pointing upward from it, orintersected the side of another filament to form T-shaped intersections.In marked contrast to resting cells, gold particles in the activatedcytoskeleton were not found as large clusters. Filament ends weregenerally decorated with one to three gold particles. Since only onegelsolin molecule is required to cap the filament end, particle groupsof one to three would therefore appear to reflect individual gelsolinmolecules. The larger clusters in the resting cytoskeletons wouldtherefore appear to reflect gelsolin clusters.

Such rearrangements of gelsolin did not occur in the Quin-2-loaded andactivated cells. Gelsolin-reactive gold in these cytoskeletons was moreclustered than in the resting cell cytoskeleton. Clusters lay on thesides of actin filaments instead of at their ends.

Example 3 Cold-induced platelet Activation

Following exposure to 4° C., actin assembly over a fifteen minute periodis about double the level of actin assembly in resting platelets andabout one third the level of actin assembly in thrombin-activatedplatelets (FIG. 4). Actin polymerization was determined by measuringphallacidin binding (Havard, T. H. and Oresajo, J., 1987 Cell Motilityand the Cytoskeleton 5:545-557).

FIG. 2 schematically illustrates the cold-induced platelet shapedistortion caused by actin filament coils. The distorted shape resemblesthat of Quin-2AM-treated platelets at room temperature (FIG. 3) and isconsistent with the hypothesis that chilling results in the uncapping,presumably ppI-mediated, of gelsolin-capped actin filament barbed end inresting platelets and desequestration of monomers. In the absence ofsevering (inhibited by the Quin-2AM treatment), filament growth resultsin actin filament coils which distort the platelet shape.

Following rewarming, the chilled platelets regain a spherical shape, achange interpreted as "recovery" many years ago by Zucker et al.(Zucker, M. B. and Borrelli, J., 1954 Blood 9:602). However, therewarmed platelets exhibit impaired hemostatic activity (Handin, R. I.et al., 1970 Transfusion 10:305; Handin, R. I. and Valeri, C. R., 1972N. Engl. J. Med. 285:538). Electron microscopic examination of theplatelets revealed that they resemble Quin-2AM-treated platelets towhich calcium was added rather than discoid (resting platelets). Theseresults suggest that chilling and rewarming results in a rise inplatelet intracellular calcium which activates gelsolin to sever thebundles formed during chilling. However, the architecture of the restingplatelet is destroyed by these events, so that subsequent activationcannot result in the normal shape changes required for optimalhemostatic activity.

Example 4 Inhibition of Cold-induced Platelet activation.

The addition of an intracellular calcium chelator (Quin-2AM) and anactin assembly inhibitor (cytochalasin B) to a platelet preparationprevented cold-induced actin bundle formation and a subsequent increasein intracellular calcium concentration.

FIGS. 6-9 illustrate the morphology of treated and/or untreated humanplatelets at 4° C. The morphologies of resting human platelets at 37° C.and cold-induced activated human platelets are shown in FIGS. 6 and 8,respectively. FIG. 7 illustrates the morphology of platelets treatedwith quin-2AM alone. FIG. 9 shows that platelets stored at 4° C. for 90minutes remain discoid only when pre-treated with both quin-2AM andcytochalasin B. These cells were identical in shape to those maintainedat 37° C. (FIG. 6).

FIG. 10 is a copy of an electron micrograph of a detergent-extracted,cold-exposed platelet which was rapidly frozen, metal-shadowed andphotographed at about 40,000 × magnification. FIG. 10 illustrates thebulbous distortion of a cold-exposed platelet by actin filament coils,which distortion resembles that observed for platelets treated withquin-2AM alone (see FIGS. 3 and 7). This result is consistent with thehypothesis that chilling results in the uncapping (presumablyppI-mediated) of gelsolin-ligated actin filament barbed ends in theresting platelets and desequestration of actin monomers. Thus, filamentgrowth, occurring in the absence of severing, results in actin filamentcoils which distort the platelet shape.

FIG. 5 illustrates actin polymerization activity in response to coldexposure or thrombin treatment (see Example 3 and Howard, T. H. andOresajo, J., 1987 supra.). The actin polymerization activity ofplatelets (never chilled) which were activated by exposure to thrombin(1 U/ml) at 37° C. is shown in FIG. 5, column E and represents thepositive control. Actin polymerization induced by chilling alone (4° C.)is shown in column A. Actin polymerization induced by chilling isenhanced for platelets that are chelated (1 uM Quin-2AM) andcytochalasin-treated (2 uM cytochalasin B) if the cytochalasin isremoved in the cold (column B). Chelation and cytochalasin treatment(treated for 30 minutes at 37° C.) completely prevented cold-inducedactin assembly so long as cytochalasin was present during the rewarmingstep (column C), even in the presence of thrombin (1 U/ml) (column D).

The actin polymerization activity of Quin-2AM-(40 uM) and cytochalasinB- (2 uM) treated platelets (treated for 90 minutes at 4° C., see FIG.5, column F), which were warmed to 37° C., washed (to removedcytochalasin B) and to which unchelated calcium (1 mM) was added,approached the actin polymerization activity observed for the positivecontrol. The morphology of the chelated and cytochalasin-treatedplatelets (FIG. 5, column E) was indistinguishable from platelets thathad never been exposed to cold temperatures.

It should be understood that the preceding is merely a detaileddescription of certain preferred embodiments. It therefore should beapparent to those skilled in the art that various modifications andequivalents can be made without departing from the spirit or scope ofthe invention.

What is claimed is:
 1. A method for the preservation of platelets withpreserved hemostatic activity, the method comprising the steps of:(a)contacting a preparation of platelets with a first agent for inhibitingactin filament severing and with a second agent for inhibiting actinpolymerization to form a treated platelet preparation; and (b) storingsaid treated platelet preparation at a temperature of less than about15° C.
 2. A method as claimed in claim 1, wherein said first agent andsaid second agent are sequentially contacted with said preparation ofplatelets.
 3. A method as claimed in claim 2, wherein said first agentis contacted with said preparation of platelets prior to contacting saidsecond agent with said preparation of platelets.
 4. A method as claimedin claim 1, wherein said first agent is an intracellular calciumchelator.
 5. A method as claimed in claim 4, wherein said intracellularcalcium chelator is a lipophilic derivative of a calcium chelatorselected from the group consisting of quin-1, quin-2, stil-1, stil-2,indo-1, fura-1, fura-2, fura-3, and BAPTA.
 6. A method as claimed inclaim 5, wherein said intracellular calcium chelator is a lipophilicderivative of quin-2.
 7. A method as claimed in claim 6, wherein saidintracellular calcium chelator is an acetoxymethyl ester of quin-2.
 8. Amethod as claimed in claim 1, wherein said second agent is selected fromthe group consisting of cytochalasin B, dihydro-cytochalasin B andcytochalasin D.
 9. A method as claimed in claim 8, wherein said secondagent is cytochalasin B.
 10. A method for making a pharmaceuticalcomposition for administration to a mammal, the method comprising thesteps of:(a) contacting a preparation of platelets contained in apharmaceutically-acceptable carrier with a first agent for inhibitingactin filament severing and with a second agent for inhibiting actinpolymerization to form a treated platelet preparation; (b) storing saidtreated platelet preparation at a temperature of less than about 15° C.to form a platelet preparation; (c) warming said platelet preparation;and (d) neutralizing said first agent and second agent in said plateletpreparation.
 11. A method as claimed in claim 10, wherein the step ofwarming said preparation of platelets is by warming the platelets to 37°C.
 12. A method as claimed in claim 10, wherein said first agent iscontacted with said preparation of platelets prior to contacting saidsecond agent with said preparation of platelets.
 13. A method as claimedin claim 10, wherein said first agent is an intracellular calciumchelator.
 14. A method as claimed in claim 13, wherein saidintracellular calcium chelator is a lipophilic derivative of a calciumchelator selected from the group consisting of quin-1, quin-2, stil-1,stil-2, indo-1, fura-1, fura-2, fura-3, and BAPTA.
 15. A method asclaimed in claim 14, wherein said intracellular calcium chelator is alipophilic derivative of quin-2.
 16. A method as claimed in claim 10,wherein said second agent is selected from the group consisting ofcytochalasin B, dihydro-cytochalasin B and cytochalasin D.
 17. A methodas claimed in claim 16, wherein said second agent is cytochalasin B. 18.A method as claimed in claim 10, wherein neutralizing said first andsecond agents is by washing said platelet preparation.
 19. A method asclaimed in claim 10, wherein neutralizing said first and second agentsis by diluting said platelet preparation with a suspension of red bloodcells.
 20. A method as claimed in claim 13, wherein neutralizing saidfirst agent is by adding unchelated calcium to said plateletpreparation.
 21. A method for mediating hemostasis in a mammal,comprising the steps of:(a) contacting a preparation of plateletscontained in a pharmaceutically-acceptable carrier with a first agentfor inhibiting actin filament severing and with a second agent forinhibiting actin polymerization to form a treated platelet preparation;(b) storing said treated platelet preparation at a temperature of lessthan about 15° C. to form a platelet preparation; (c) warming theplatelet preparation; (d) neutralizing said first and second agents insaid platelet preparation to form a pharmaceutical composition; and (e)administering said pharmaceutical composition to the mammal.
 22. Amethod as claimed in claim 21, wherein the step of warming saidplatelets is by warming the platelets to 37° C.
 23. A method as claimedin claim 22, wherein said first agent is contacted with said preparationof platelets prior to contacting said second agent with said preparationof platelets.
 24. A method as claimed in claim 21, wherein said firstagent is an intracellular calcium chelator.
 25. A method as claimed inclaim 24, wherein said intracellular calcium chelator is a lipophilicderivative of a calcium chelator selected from the group consisting ofquin-1, quin-2, stil-1, stil-2, indo-1, fura-1, fura-2, fura-3, andBAPTA.
 26. A method as claimed in claim 25, wherein said intracellularcalcium chelator is a lipophilic derivative of quin-2.
 27. A method asclaimed in claim 21, wherein said second agent is selected from thegroup consisting of cytochalasin B, dihydro-cytochalasin B andcytochalasin D.
 28. A method as claimed in claim 27, wherein said secondagent is cytochalasin B.
 29. A method as claimed in claim 21, whereinneutralizing said first and second agents is by washing said plateletpreparation.
 30. A method as claimed in claim 29, wherein neutralizingsaid first and second agents is by diluting said platelet preparationwith a suspension of red blood cells.
 31. A method as claimed in claim21, wherein neutralizing at least one of said first agent and saidsecond agent is by removing at least one of said first agent and saidsecond agent from said platelet preparation.
 32. A method as claimed inclaim 24, wherein neutralizing said first agent is by adding unchelatedcalcium to said platelet preparation.